System and Method for Normalized Flourescence or Bioluminescence Imaging

ABSTRACT

A system and method provide normalized fluorescence epi-illumination images and normalized fluorescence transillumination images. The normalization can be used to improve two-dimensional (planar) fluorescence epi-illumination images and two-dimensional (planar) fluorescence transillumination images. The system and method can also provide normalized bioluminescence epi-illumination images and normalized bioluminescence transillumination images. In some arrangements, the system and method can provide imagine of small animals, into-operative imaging, endoscopic imaging, and/or imaging of hollow organs.

FIELD OF THE INVENTION

This invention relates generally to medical imaging and, moreparticularly, to a system and method for generating fluorescenceepi-illumination images, fluorescence transillumination images, andbioluminescence images associated with small animal imaging,intra-operative imaging, endoscopic imaging and imaging of holloworgans.

BACKGROUND OF THE INVENTION

Fluorescence images can be generated in-vivo for imaging of molecularfunctions and gene expression in live biological tissues. Fluorescenceimaging of small animals has been used in biological research, includingresearch in drug discovery and in investigation of fluorescent reportertechnologies. Fluorescence imaging has also been used to study varioushuman tissues, for example, tissues exhibiting epithelial diseases, thehuman breast, joints, and human teeth.

Conventionally, fluorescent light has been used for high-resolutionimaging of histological slices of biological tissue using so-calledfluorescence microscopy. Fluorescence microscopy is used to providerelatively high-resolution images. However, tissue sectioning used inconventional fluorescence microscopy is limited to slice thicknesses(i.e., tissue depths) on the order of half a millimeter, and therefore,conventional fluorescence microscopy is not appropriate for imagingthrough entire organs or through the whole human body.

In order to provide images a bit deeper into tissue, conventionalsystems and techniques have used light sources and fluorochromes thatemit near infrared light. The near infrared light is selected becausenear infrared light has low absorption and can penetrate severalcentimeters into biological tissue. Near infrared light is used in avariety of optical imaging systems and techniques.

Fluorescent light can be emitted from a tissue in response to anexcitation light source transmitting excitation light into the tissue.The excitation light excites the emission of fluorescent light fromfluorochromes within the tissue.

Similarly, bioluminescence imaging has been used to image into tissues.The difference between fluorescence and bioluminescence imaging is that,for bioluminescence imaging, no excitation light source is required tocause emission of bioluminescent light. Emission of bioluminescent lightin bioluminescence imaging is caused by a chemiluminescent reactionwithin the tissue, resulting from transgenes.

The most common macroscopic technique that is conventionally used forfluorescence imaging is fluorescence reflectance imaging (FRI), which isalso referred to herein as fluorescence epi-illumination imaging (FET).

Epi-illumination light sources and epi-illumination imaging are furtherdescribed below. In general, an epi-illumination light source generateslight that is directed toward and then reflects from a surface ofbiological tissue and/or that propagates into the biological tissue andreflects from internal structures and/or surfaces of the biologicaltissue. To form an epi-illumination image, image light is collectedgenerally on the same side of the tissue as the epi-illumination lightsource.

An FEI system transmits light onto and/or into biological tissue andcollects the fluorescence light that is emitted back from the tissue,including light that is emitted back from within the tissue. Influorescence epi-illumination imaging, excitation light (for example,near-infrared light) from an epi-illumination light source is used toilluminate the tissue. The epi-illumination light source is used toexcite fluorochromes within the tissue that, in turn, emit fluorescentlight. In some arrangements, the emitted light is visible light. Inother arrangements, the emitted light is near infra red light. Theemitted light can be visually inspected or it can be captured with a CCDcamera or other photon detector positioned generally on the same side ofthe tissue as the epi-illumination light source. Bioluminescence imagingcan be similar to fluorescent epi-illumination imaging, butbioluminescence is generated without an epi-illumination light source.

As described above, conventional fluorescence imaging with near-infraredlight provides images having relatively low resolution and only smallpenetration (2-3 mm) of tissue. Higher resolution is achieved whenspectral information is utilized and “mixed.”

A second method, which has not yet been utilized for research usingsmall animals, but which has found applications in optical breastimaging, uses a transillumination light source to generatetransillumination images. Similar to the above-describedepi-illumination light source, a transillumination light sourcegenerates light that propagates into the tissue. However, unlikeepi-illumination light, the transillumination light propagates entirelythrough the tissue. In transillumination imaging, image light iscollected generally on the opposite side of the tissue from thetransillumination light source.

Similar to that described above for fluorescence epi-illuminationimaging, in fluorescence transillumination imaging, excitation light(for example, near infra red light) from a transillumination lightsource is used to illuminate a tissue. The excitation light propagatesinto the tissue, exciting the emission of fluorescent light from withinthe tissue. However, in contrast to the above-described fluorescenceepi-illumination arrangement, in fluorescence transillumination imaging,a CCD camera or other photon detector is positioned generally on theopposite side of the tissue from the transillumination light source. Insome arrangements, the emitted light is near infrared light.Fluorescence transillumination imaging (FTD) has been used to visualizefunctional characteristics of cardiac muscle and in dental diagnosticpractice.

In some transillumination arrangements, the transillumination lightsource and the light detector lie on a virtual line passing through thetissue. In some arrangements the virtual line is generally perpendicularto the tissue and, in other arrangements, the virtual line is notgenerally perpendicular to the tissue.

Fluorescence epi-illumination imaging (FEI), fluorescencetransillumination imaging (FTI), and bioluminescence imaging (BD) areforms of “planar” imaging, which provide two-dimensional images.

More advanced optical imaging systems and methods have been developed,which utilize tomographic methods. These systems and methods operate byobtaining photonic measurements at different projections (i.e., angles)to the tissue and combining the measurements using a tomographicalgorithm. Tomography can provide a more accurate image than theabove-described forms of planar imaging. Advantages of tomographyinclude an ability for image qualification, an ability to providetwo-dimensional or three-dimensional images, an ability to providethree-dimensional imaging with feature depth measurements, and highersensitivity and higher resolution as compared to planar imaging. In someapplications, tomography has been used in-vivo to measure enzymeregulation and treatment response to drugs. In these applications,tomography provides superior imaging performance to planar imaging.However, tomography is more complex than planar imaging, requiring moreadvanced instrumentation, requiring multiple illumination points(projections), which can require multiple light sources, and requiringadvanced theoretical methods for modeling photon propagation in tissues.

SUMMARY OF THE INVENTION

The system and method for normalized epi-illumination imaging andnormalized transillumination imaging provide normalization of imagesgenerated by planar fluorescence epi-illumination imaging, by planarfluorescence transillumination imaging, by planar bioluminescenceepi-illumination imaging, and by planar bioluminescencetransillumination imaging. The normalization results in substantiallyimproved images. As further described below, by using a combination ofnormalized epi-illumination images and normalized transilluminationimages, each having particular imaging characteristics, still furtherimprovement can be achieved.

In some particular arrangements, as further described below, the systemand method can be used during surgery to identify tumors or otherlesions and borders thereof. In other words, the system and method canbe used for intra-operative imaging. In some arrangements, the systemand method can also be used for fluorescence endoscopy and/orlaparoscopy. In some arrangements, the system and method can also beused in oral and dental procedures to provide oral or dental images.

In accordance with the present invention, a method of imaging includesgenerating incident light including excitation light with an excitationlight source and directing the incident light toward a tissue. Themethod also includes receiving the incident light with a light detectorafter the incident light has interacted with a tissue. The methodfurther includes receiving emitted light with the light detector,wherein the emitted light is emitted from the tissue. The method furtherincludes generating an intrinsic image of the tissue in response to theincident light. The method further includes generating an un-normalizedemitted light image of the tissue. The method further includes combiningthe uni-normalized emitted light image and the itrintsic image togenerate a normalized emitted light image of the tissue.

The normalized emitted light image generated by the method is anormalized fluorescence epi-illumination image, a normalizedfluorescence transillumination image, a normalized bioluminescenceepi-illumination image, and/or a normalized bioluminescencetransillumination image.

In accordance with another aspect of the present invention, a system forimaging a tissue includes an excitation light source adapted to generateincident light including excitation light. The system also includes alight receiver adapted to receive the incident light after the incidentlight has interacted with a tissue, further adapted to receive emittedlight, wherein the emitted light is emitted from the tissue, furtheradapted to generate an intrinsic image of the tissue in response to theincident light, and further adapted to generate an un-normalized emittedlight image of the tissue. The system further includes a normalizationprocessor adapted to combine the un-normalized emitted light image andthe intrinsic image to generate a normalized emitted light imageassociated with the tissue.

The normalized emitted light image generated by the system is anormalized fluorescence epi-illumination image, a normalizedfluorescence transillumination image, a normalized bioluminescenceepi-illumination image, and/or a normalized bioluminescencetransillumination image.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features of the invention, as well as the invention itselfmay be more fully understood from the following detailed description ofthe drawings, in which:

FIG. 1 is a pictorial of system used for normalized fluorescenceepi-illumination imaging and normalized fluorescence transilluminationimaging having a normalization processor;

FIG. 1A is a pictorial showing two light masks that can be used inconjunction with light sources of FIG. 1;

FIG. 1B is a pictorial of another system used for normalizedfluorescence epi-illumination imaging and normalized fluorescencetransillumination imaging having another normalization processor;

FIG. 1C is a block diagram showing further details of the normalizationprocessors of FIGS. 1 and 1B;

FIG. 2 is a pictorial showing greater detail of a light detector thatcan be used in the normalized fluorescence epi-illumination imaging andnormalized fluorescence transillumination imaging systems of FIGS. 1 and1B;

FIG. 2A is a pictorial showing greater detail of another light detectorthat can be used in the normalized fluorescence epi-illumination imagingand normalized fluorescence transillumination imaging systems of FIGS. 1and 1B;

FIG. 3 is a series of images of a phantom, generated by the systems ofFIGS. 1 and 1B, when used for normalized fluorescence epi-illuminationimaging;

FIG. 3A is a series of images of the phantom, also seen in FIG. 3,provided by the systems of FIGS. 1 and 1B when used for normalizedfluorescence transillumination imaging;

FIG. 4 is a series of images of the phantom, also seen in FIGS. 3 and3A, showing an intrinsic transillumination image at an excitation lightwavelength, a fluorescence transillumination image, and a normalizedfluorescence transillumination image;

FIG. 5 is a series of images of a phantom showing an intrinsicepi-illumination image at the excitation light wavelength, afluorescence epi-illumination image, and a normalized fluorescenceepi-illumination image;

FIG. 5A is another series of images of another phantom, showing anintrinsic epi-illumination image at the excitation light wavelength, afluorescence epi-illumination image, and a normalized fluorescenceepi-illumination image;

FIG. 5B is a series of images of the phantom, also seen in FIG. 5,showing an intrinsic transillumination image at the excitation lightwavelength, a fluorescence transillumination image, and a normalizedfluorescence transillumination image;

FIG. 5C is another series of images of the phantom, also seen in FIG.5A, showing an intrinsic transillumination image at the excitation lightwavelength, a fluorescence transillumination image, and a normalizedfluorescence transillumination image;

FIG. 6 is a series of images of a mouse showing an intrinsicepi-illumination image at the excitation light wavelength, afluorescence epi-illumination image, and a normalized fluorescenceepi-illumination image;

FIG. 6A is a series of images of the mouse of FIG. 6 showing anintrinsic transillumination image at the excitation light wavelength, afluorescence transillumination, and a normalized fluorescencetransillumination image;

FIG. 7 is a series of images of another mouse showing an intrinsicepi-illumination image at the excitation light wavelength, afluorescence epi-illumination image, and a normalized fluorescenceepi-illumination image;

FIG. 7A is a series of images of the mouse of FIG. 7 showing anintrinsic transillumination image at the excitation light wavelength, afluorescence transillumination image, and a normalized fluorescencetransillumination image;

FIG. 8 is a series of images showing an intrinsic epi-illumination lightimage at the excitation wavelength, a fluorescence epi-illuminationimage, a normalized fluorescence epi-illumination image, and anormalized fluorescence epi-illumination image after the application ofnoise thresholds, showing detection of tumors;

FIG. 8A is a series of images showing an intrinsic epi-illuminationimage at the excitation light wavelength, a fluorescenceepi-illumination image, a normalized fluorescence epi-illuminationimage, and a normalized fluorescence epi-illumination image after theapplication of noise thresholds, showing detection of tumors;

FIG. 9 is a pictorial of a system used for intra-operative imaging,including a portable camera/illuminator combination, a normalizationprocessor, and a monitor, and, in some embodiments, a flexible probe(illuminator/laparoscope/endoscope);

FIG. 9A is a pictorial of another system used for intra-operativeimaging, including goggles that can be worn by a surgeon;

FIG. 10 is a pictorial of a system that can be used for dental imaging;

FIG. 11 is a pictorial of a system used for colposcopy having ancolposcopic probe; and

FIG. 11A is a pictorial showing greater detail of a face of thecolposcopic probe of FIG. 11.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the imaging system and method, some introductoryconcepts and terminology are explained. As used herein, the term“phantom” is used to describe a test object being imaged. A phantom istypically an article having diffuse light propagation characteristicssimilar to living tissue, for example, a piece of appropriatelyengineered resin block. For another example, a phantom can be a vial,which contains cells having fluorescent proteins therein, i.e. afluorescent marker or a fluorochrome.

As used herein, the term “excitation light” is used to describe lightgenerated by an “excitation light source” that is incident upon abiological tissue. The excitation light can interact with the tissue,and can be received by a light detector (e.g., a camera), at the samewavelength (excitation wavelength) as that at which it was transmittedby the excitation light source. The excitation light can bemonochromatic, or it can cover a broader spectrum, for example, whitelight. The excitation light can be used to generate a so-called“itrinsic excitation light image” of the tissue (or more simply, andintrinsic image), i.e., an image obtained at the same wavelength as thewavelength of the excitation light. The excitation light can also beused to excite fluorescence within the tissue, and to generate aso-called “fluorescence image” of the tissue, at a selected differentwavelength than the wavelength of the excitation light.

As used herein, the term “incident light” is used to more generallydescribe light that is generated by the excitation light source, whereinthe incident light can include not only the excitation light having awavelength selected to excite fluorescence, but also light having otherwavelengths. The incident light can include only the excitation light,or it can also include other wavelengths. The wavelengths of theincident light can be generated simultaneously or at different times.The incident light can be used to generate a so-called “intrinsicincident light image” of the tissue (or more simply, an intrinsicimage). The intrinsic incident light image is either an image obtainedat the same wavelength as the wavelength of the excitation light (i.e.,an intrinsic excitation light image), an image obtained at a differentwavelength than the wavelength of the excitation light, or an imageobtained by a combination of images obtained at a variety ofwavelengths, which may or may not include the wavelength of theexcitation light. The combination of images is further described below.

In general, it will be understood that an intrinsic image is an image ofnatural structures inside the tissue, exclusive of any fluorescence ofthe tissue or of fluorescence generated by fluorescence markers withinthe tissue. In contrast, a fluorescence image is an image of only thetissue fluorescence or of fluorescence generated by the fluorescencemarkers within the tissue. The intrinsic image can be either anintrinsic excitation light image, or, more generally, an intrinsicincident light image.

As used herein, the term “epi-illumination light source” is used todescribe an excitation light source that can generate a form ofexcitation light (also referred to herein as “epi-illumination light”)that reflects from a surface of biological tissue and/or that propagatesinto the biological tissue, in order to form a so-called“epi-illumination image.” To form an epi-illumination image, image lightis collected generally on the same side of the tissue as theepi-illumination light source. An epi-illumination image can be eitheran intrinsic epi-illumination image (no fluorescence) or a fluorescenceepi-illumination image (only fluorescence).

As used herein, the term “transillumination light source” is used todescribe an excitation light source that can generate a form ofexcitation light (also referred to herein as “transillumination light”)that propagates into the tissue, in order to generate a so-called“transillumination image.” To form a transillumination image, light iscollected generally on the opposite side of the tissue from thetransillumination light source. Like an epi-illumination image, atransillumination image can be either an intrinsic transilluminationimage (no fluorescence) or a fluorescence transillumination image (onlyfluorescence).

In some arrangements, the epi-illumination light source and/or thetransillumination light source(s) can, more generally, generate incidentlight, which includes the excitation light.

To generate an intrinsic epi-illumination image, the excitation light(epi-illumination light) is received by a camera after being directedback from (e.g., reflected from) an object being imaged. In contrast, togenerate an intrinsic transillumination image, the excitation light(transillumination light) is be received by a camera after it passesthrough the object being imaged.

Similarly, to generate a fluorescence epi-illumination image, theexcitation light (epi-illumination light) excites fluorescence on or inthe tissue, which is directed back from tissue being imaged, and whichis received at a wavelength different from the excitation light. Togenerate a fluorescence transillumination image, the excitation light(transillumination light) also excites fluorescence in the tissue, whichis directed through the tissue being imaged, and which is received at awavelength different from the excitation light.

As used herein, the term “emitted light” is used to describe lightgenerated by or within a biological tissue. As used herein, the term“fluorescence” is used to describe a form of emitted light generated viaexcitation of a marker fluorochrome in response to the excitation light.As used herein, the term “bioluminescence” is used to describe anotherform of emitted light emitted from a tissue, generally in the absence ofthe excitation light. As used herein, the term “emitted light image” isused to describe either a fluorescence image or a bioluminescence image.

As used herein, the term “image” is used to describe a visualpresentation of something such as an object or a scene. An image may berepresented as image data, which can be displayed on a computer monitor.Image data can be generated by a digital camera or other image deviceand provided to a computer system or other processing device. However,it will be understood that the term “image,” as used herein, is alsoused to refer to the image data.

The system and method described herein to explain inventive conceptsrefer to the use of particular types of light or light having particularcharacteristics. For example, reference is made to systems and methodsusing near-infrared excitation light, which provides particular benefitsin the near-infrared (NIR) wavelength range of about 650-1000 nm. Itshould, however, also be appreciate that the system and method describedherein can also be applied to excitation light having other wavelengths,for example to light in the visible range of about 400 nm-650 nm. Also,the system and method apply to a system in which excitation light isgenerated by an excitation light source in one wavelength range, forexample, in the visible range, and the fluorescent light emitted byfluorochromes is in another wavelength range, for example in the NIRrange. The system and method also apply where both the excitation lightgenerated by the excitation light source and the light emitted by thefluorochromes are in the NIR range. In addition, excitation light and/oremitted light having a wavelength at the interface of visible range andnear-infrared range can be used, for example in the 550 nm-650 nm range.Some wavelengths are particularly beneficial for bioluminescence imagingand for imaging of red-shifted fluorochromes and fluorescent proteins.The excitation light can have the same intensity at all wavelengthstherein, or it can have predetermined attenuation of selectedwavelengths, e.g., by using appropriate filters. Also, excitation lightbeyond the wavelength range of 400 nm to 1000 nm can be used.

The term “fluorochrome” as used herein will be understood to describe atype of known biocompatible dye or other fluorescence agent that can besystemically or topically applied to a biological tissue. Somefluorochromes are target fluorochromes, which tend to congregate atcertain anatomical, functional, or molecular features in the tissue,including for example, at cancerous lesions.

Referring now to FIG. 1, an imaging system 10 includes a plurality oftransillumination light sources 12 adapted to generate excitation(transillumination) light, which is represented by a plurality of arrows14. The system 10 also includes an epi-illumination light source 16adapted to generate excitation (epi-illumination) light represented byan arrow 18 and lines 18 a, 18 b, which are generally indicative oflight beam boundaries. Thus, the epi-illumination light source 16 emitsa single relatively broad beam of light 18, 18 a, 18 b. Thetransillumination light 14 and the epi-illumination light 18, 18 a, 18 bare directed toward a biological tissue 24. In this particular example,the biological tissue is shown to be a mouse 24.

In some embodiments, an optional light mask 26 can be disposed betweenthe transillumination light sources 12 and the biological tissue 24.Thus, in those embodiments, which utilize a light mask such as lightmask 26, the transillumination light 14 passes through the light mask26, and impinges upon the biological tissue 24. The light mask isfurther described below in greater detail in conjunction with FIG. 1A.

The system 10 also includes a camera 20 (or other type of detector)adapted to receive light represented by lines 22 a, 22 b, which linesare generally indicative of light beam boundaries. As further describedbelow, the light 22 a, 22 b is associated with the transilluminationlight sources 12 and/or with the epi-illumination light source 16. Thecamera 20 generates image information 28, which is received by anormalization processor 30. The normalization processor 30 generatesnormalized image information 32, which is received by and displayed by adisplay 34. The normalization processor 30 is further described below,where it will become apparent that the normalization provided by thenormalization processor 300 results in improved “normalized”fluorescence images of a subject 24.

In operation, the camera 16 receives the light 26 a, 26 b. In someembodiments, the transillumination light sources 18 and theepi-illumination light source 16 generate excitation light, 18, 20,respectively, concurrently and the camera 16 receives light 26 a, 26 bhaving contributions from both the transillumination light sources 12and the epi-illumination light source 16 at the same wavelength as eachof the excitation light sources 12, 16. In other embodiments, thetransillumination light sources 12 and the epi-illumination light source16 generate the excitation light 18, 20, respectively, at differenttimes, so that at any particular time the camera 16 receives the light26 a, 26 b from one of the transillumination light sources 12 and theepi-illumination light source 16.

The light 26 a, 26 b received by the camera 16 in response to theexcitation light 18, 18 a, 18 b generated by the epi-illumination lightsource 16 can have contributions from the excitation light 18, 18 a, 18b at the excitation light wavelength and also contributions from emittedlight (fluorescence or bioluminescence), emitted within the subject 24at a different wavelength in response to the excitation light 18, 18 a,18 b. Contributions from the excitation light 18, 18 a, 18 b can be usedto form an intrinsic epi-illumination image, and contributions from theresulting emitted fluorescent light can be used to form a fluorescenceepi-illumination image and also a normalized fluorescenceepi-illumination image.

Similarly, the light 26 a, 26 b received by the camera 16 in response tothe excitation light 14 generated by the transillumination light source12 can have contributions from the excitation light 14 at the excitationlight wavelength and contributions from emitted light (fluorescence orbioluminescence), emitted within the subject 24 at a differentwavelength in response to the excitation light 14. Contributions fromthe excitation light 14 can be used to form an intrinsictransillumination image, and contributions from the emitted light can beused to form a fluorescence transillumination image and also anormalized fluorescence transillumination.

From the above discussion, it should be recognized that the system 10can generate normalized fluorescence epi-illumination images, normalizedfluorescence transillumination images, or both normalized fluorescenceepi-illumination images and normalized fluorescence transilluminationimages. The system 10 can also generate normalized bioluminescenceimages.

As described above, normalized fluorescence epi-illumination images canbe generated by illuminating tissue (e.g. the mouse 24 in FIG. 1) withthe excitation light 18, 18 a, 18 b generated by the epi-illuminationlight source 16. The camera 20, positioned on the same side of thetissue 24 as the epi-illumination light source 16, is used to captureboth excitation (epi-illumination) light reflected from the tissue 24 toform an intrinsic epi-illumination image, and fluorescent light, whichis emitted from within the tissue 24 in response to the excitation(epi-illumination) light 18, 18 a, 18 b to form an un-normalizedfluorescence epi-illumination image. The intrinsic epi-illuminationimage and the un-normalized fluorescence epi-illumination image arecombined by the normalization processor 30 to generate the normalizedfluorescence epi-illumination image.

Also as described above, normalized fluorescence transilluminationimages can be generated by illuminating the tissue (here the mouse 24)with the excitation light 14 generated by the transillumination lightsources 12. The camera 20, positioned on the opposite side of the tissue24 as the transillumination light sources 12, is used to capture bothexcitation (transillumination) light passing through the tissue 24 togenerate an intrinsic transillumination image, and fluorescent light,which is emitted from within the tissue 24 in response to the excitation(transillumination) light 14 to form an un-normalized fluorescencetransillumination image. The intrinsic transillumination image and theun-normalized fluorescence transillumination image are combined by thenormalization processor 30 to generate the normalized fluorescencetransillumination image.

To generate normalized bioluminescence images, the system 10 collects anun-normalized image of bioluminescence light. In this case,bioluminescence light is spontaneously emitted and is not generated inresponse to the excitation lights 14, 18, 18 a, 18 b. The un-normalizedbioluminescence image can be combined by the normalization processor 30with either the intrinsic epi-illumination image or with the intrinsictransillumination image to generate the normalized bioluminescenceimage.

For either epi-illumination imaging or transillumination imaging, inorder to form the intrinsic images in some embodiments, the excitationlight source generates one or more wavelengths (i.e. incident light)including, but not limited to, a wavelength of excitation light, and theintrinsic image is generated by combining intrinsic images associatedwith the one or more wavelengths. In some arrangements the combinationis a weighted average.

While the term “excitation light” is described above to be associatedwith light that can excite fluorescence in biological tissue, theexcitation light can also be used by the system 10 in order to generatethe normalized bioluminescence images. In some arrangements, theexcitation light can be generated at the bioluminescence wavelengths sothat it can be used to capture the propagation characteristics of thebioluminescence in the tissue. For example, as described above, innormalized bioluminescence transillumination imaging, the system 10collects both an image of the excitation (transillumination) light(intrinsic image) and an un-normalized image of bioluminescence light.

As described above, normalized fluorescence transillumination imaging isprovided by positioning the plurality of transillumination light sources12 generally on the other side of the tissue 24 from the camera 20. Thetransillumination light sources 12 can have an illumination pattern, forexample, by using an array of transillumination light sources 12disposed in a predetermined pattern, or by using an optical guidingsystem disposed in the predetermined pattern, for example, the lightmask 26

In fluorescence transillumination imaging using the plurality oftransillumination light sources 12, transillumination imaging can beaccomplished by a superposition of light signals associated withexcitation light generated by each one of the plurality oftransillumination light sources 12. In essence, each one of thetransillumination light sources 12 can be sequentially illuminated andresulting intrinsic and fluorescence images can be sequentiallycaptured.

The camera 20 can collect both excitation light associated with thelight sources 12, 16, which is reflected from and/or which passedthrough the tissue 24, and can collect emitted light associated with afluorochrome within the tissue and/or associated with an endogenoustissue fluorescent molecule. The system 10 can also collect emittedbioluminescence light, for example, when the excitation light sources12, 16 are turned off.

The same system 10, using the same camera 20, can be used to generateboth normalized fluorescence epi-illumination images and normalizedfluorescence transillumination images. The camera 20 is shown in greaterdetail in conjunction with FIGS. 2 and 2A.

While it is described above that the epi-illumination light source 16and the transillumination light sources 12 are adapted to generateexcitation light, in other arrangements, the epi-illumination lightsource 16 and/or the transillumination light sources 12 are adapted togenerate incident light having the wavelength of the excitation lightand also other wavelengths.

Referring now to FIG. 1A which is comprised of a first panel labeled“(a)” and a second panel labeled “(b)”, a first exemplary light mask 26a shown in the panel (a) and a second exemplary light mask 26 b shown inthe panel (b). The masks 26 a, 26 b serve to block stray light and alsoexcitation light that could otherwise directly hit the camera (or otherdetector) and saturate it or damage it. Additional absorbing materialscan be used to selectively prevent excitation light 14, 18 generated bythe excitation light sources 12, 16, respectively, from directlyentering the camera 20. In other arrangements, selective beam-scanpatterns can be used to avoid direct exposure of the camera 20 toexcitation light 14, 18. In other arrangements, adaptive attenuation oflight intensity from the plurality of transillumination light sources 12can be used to prevent saturation of the camera 20 and also to improvethe dynamic range of images acquired by the camera 20. For example,dynamic range can be improved for light that is minimally attenuated bytissue, for example, light close to the tissue borders.

Referring flow to FIG. 1B, another system 50, which is similar to thesystem 10 of FIG. 1, but which includes a single transillumination lightsource 52 having a relatively wide beam of excitation light, representedby an arrow 54, and by lines 54 a, 54 b, which are generally indicativeof light beam boundaries. In other arrangements, the emitted light 54,54 a, 54 b could have a spatially attenuated pattern to prevent CCDpixel saturation close to and outside of borders of tissue 64.

Other elements of FIG. 1B can be the same as or similar to elements ofFIG. 1. For example, d mask 66 can be the same as or similar to the mask26, an epi-illumination light source 56 can be the same as or similar tothe epi-illumination light source 16, a camera 60 can be the same as orsimilar to the camera 20, a normalization processor 70 can be the sameas or similar to the normalization processor 30, and a display 74 can bethe same as or similar to the display 34.

For both the system 10 of FIG. 1 and the system 50 of FIG. 1B, methodsof generating “normalized” images are described below using“un-normalized” images. Generation of a normalized fluorescenceepi-illumination images involves collection of at least two separateimages (i.e., two types of image data) that are combined to provide thenormalized fluorescence epi-illumination image (or normalized imagedata). The two images include an image of the tissue (e.g., 24, FIG. 1)resulting from epi-illumination excitation light (e.g., 18, 18 a, 18 b,FIG. 1) generated by an epi-illumination light source (e.g., 16, FIG.1), which is generally reflected from the tissue 24 (FIG. 1), and alsoan un-normalized image of emitted light from a fluorescent probe (i.e.,a fluorochrome). The emitted light is excited by the excitation light18, 18 a, 18 b. The image of the excitation light 18, 18 a, 18 b is alsoreferred to herein as an intrinsic epi-illumination image and the imageof the emitted light is also referred to herein as an un-normalizedfluorescence epi-illumination image.

In some embodiments, two dark light images (i.e., background images thatcan contain, for example, stray light and noise) are also collected andused in combination with the above two images to generate the normalizedfluorescence epi-illumination image. One dark light image can begenerated using the same acquisition settings (gain, exposure time,etc.), which are used to capture the above-described intrinsicepi-illumination image, and the other dark light image can be generatedusing the same acquisition settings, which are used to capture theabove-described un-normalized fluorescence epi-illumination image. Useof dark images will become apparent from equations below.

Similarly, generation of a normalized fluorescence transilluminationimage involves collection of at least two separate un-normalized images(i.e., two types of image data) that are combined to provide thenormalized fluorescence transillumination image (or normalized imagedata). The two un-normalized images include an image of the tissue(e.g., tissue 24, FIG. 1) generated from transillumination excitationlight (e.g., excitation light 14, FIG. 1) generated by atransillumination light source (e.g., source 12, FIG. 1), which passesthrough the tissue 24, and an image of emitted light from a fluorescentprobe (i.e., a fluorochrome). The emitted light is excited by theexcitation light 14. The image of the excitation 14 is also referred toherein as an intrinsic transillumination image and the image of theemitted light is also referred to herein as an un-normalizedfluorescence transillumination image.

In some embodiments, two dark light images are also collected and usedto generate the normalized fluorescence transillumination image. Onedark light image can be generated using the same acquisition settings(gain, exposure time, etc.), which are used to capture theabove-described intrinsic transillumination image, and the other darklight image can be generated using the same acquisition settings, whichare used to capture the above-described un-normalized fluorescencetransillumination image.

Similarly, for bioluminescence, at least two, but in some embodimentsfour, images can collected, i.e. the intrinsic image and dark image forexcitation light (epi-illuminated or transilluminated) and the emittedimage and dark image for bioluminescence.

For embodiments using dark images, the dark images need not be acquiredin each measurement but can be acquired once and stored in memory. Asdescribed above, in some embodiments, the dark current images may not beused, especially if dark images have very low contrast. In some otherembodiments, an equivalent subtraction method may be used.

For both normalized fluorescence epi-illumination imaging and normalizedfluorescence transillumination imaging, the excitation light provided bythe light source and the emitted light generated by the fluorescentprobe can be at different wavelengths. The emitted light received togenerate bioluminescence images and the excitation light employed inassociated intrinsic light images can be at different wavelengths or atthe same wavelength. In one particular embodiment, the emitted light isnear infrared light and the excitation light is also near infrared lightbut at a shorter wavelength. In one particular embodiment ofbioluminescence imaging, the emitted light contains visible andnear-infrared spectral components and the excitation light containssimilar spectral components.

The following equations can be used in a method to generate normalizedand noise-reduced fluorescence epi-illumination images and normalizedand noise-reduced transillumination fluorescence images. Such a methodcan be performed in a system similar to systems 10, 50 described abovein conjunction with FIGS. 1 and 1B, respectively. It will be understoodthat, in some embodiments, the un-normalized images described below canbe generated by the camera 20 of FIG. 1 or the camera 60 of FIG. 1B. Thenormalized images described below (and also the noise reducedun-normalized images), which can be generated by further processingand/or combination of the un-normalized images, can be generated by thenormalization processor 30 of FIG. 1 or the normalization processor 70of FIG. 1B.

An un-normalized and noise-reduced fluorescence epi-illumination image,Ie, can be expressed as:

I _(e) =I _(fe) −I _(fn)  (Eq. 1)

where I_(fe) is the fluorescence image generated when using anepi-illumination light source (e.g. light source 16, FIG. 1) and I_(fn)is the background camera noise image (i.e., a dark image or a constant),respectively. I_(fn) can be acquired with identical acquisition andexperimental parameters as I_(fe) but in the absence of excitationlight, or it can be approximated by an equivalent constant value, whichcan be based on an experimental measurement. Hereafter, the image,I_(e), is referred to as an un-normalized noise-reduced fluorescenceepi-illumination image, or more simply an un-normalized fluorescenceepi-illumination image, where the noise is presumed.

Similarly an un-normalized and noise-reduced fluorescencetransillumination image, I_(f), can be expressed as:

$\begin{matrix}{I_{t} = {\sum\limits_{k = 1}^{Ns}{{g(k)}\left( {{I_{f\; t}(k)} - I_{fn}} \right)_{> {Tf}}}}} & \left( {{Eq}.\mspace{14mu} 2} \right)\end{matrix}$

where Ns is the number of back-illuminating transillumination lightsources (e.g., light sources 12, FIG. 1) where Ns≧1), I_(ft)(k) is afluorescence transillumination image obtained by illuminating the k^(th)transillumination light source, and I_(fn) is a corresponding noiseimage (also including an offset) obtained under identical conditions butwith no illumination by an excitation light source (i.e., a dark image).The factor g(k) is a percentage coefficient that is included to correctfor the variation of individual transillumination light source strengthscompared to a median source strength as calculated based on the relativestrength of each source, which values are measured, for example, througha homogenous intralipid. Tf is a threshold value.

All fluorescence image values above the threshold, Tf, are summedtogether (superimposed) to yield the un-normalized noise-reducedfluorescence transillumination image. In one particular embodiment, thethreshold, Tf, is set at 10 times the standard deviation of the photoncounts seen in the I_(fn) image. The value of the threshold, Tf, isselected to prevent noise, or to prevent image signals having a lowsignal to noise ratio from being included in the un-normalizedfluorescence transillumination image, I_(t).

Hereafter, the image, I_(t), is referred to as an un-normalizednoise-reduced fluorescence transillumination image, or more simply anun-normalized fluorescence transillumination image, where the noisesubtraction may be presumed.

The above un-normalized fluorescence epi-illumination image, Ie, and theun-normalized fluorescence transillumination image, I_(t), can benormalized in the following way. The normalization process divides theun-normalized images by corresponding intrinsic images (i.e. by an imageof epi-illumination excitation light for epi-illumination imaging or byan image of transillumination excitation light for transilluminationimaging).

A normalized fluorescence epi-illumination image, U_(e), can be computedas:

$\begin{matrix}{U_{c} = \frac{{a \cdot \left( {I_{fe} - I_{fn}} \right)_{> {Tf}}} + c_{f}}{{b \cdot \left( {I_{ee} - I_{en}} \right)_{> {Te}}} + c_{e}}} & \left( {{Eq}.\mspace{14mu} 3} \right)\end{matrix}$

where I_(ee) is a epi-illumination image obtained at the excitationlight wavelength (i.e., an intrinsic epi-illumination image, which is animage of the tissue generated from excitation light generated by aepi-illumination light source having reflected from the tissue) andI_(en) is camera noise and offset obtained with no excitation from thelight source (or dark image).

Normalized image performance depends upon the threshold values Tf (forthe fluorescence epi-illumination image) and Te (for the intrinsicepi-illumination image). In one particular embodiment, Ue is set to zerounless the I_(ee)-I_(en) denominator values are above the threshold, Te,and the I_(fe)-I_(fn), values are above the threshold, Tf. Selection ofthe thresholds can be based upon noise statistics or empirical data. Theselection can be made statically by selecting values a predeterminednumber of noise standard deviations of the noise image (for example, 20standard deviations of the noise image, I_(en)), or adaptively. Theconstants c_(f) and c_(e) can also be statically or adaptively selected,and their purpose is to stabilize the image. The constants c_(f) andc_(e) can have a value of zero, depending on user preferences, or theymay be small values to provide small offset values to stabilize theratio and yield a more accurate visual result. In one particularembodiment, a default setting of cf=0 and c_(e)=1 is used to avoiddivision by zero. Similarly, factors a and b can implement a-prioriinformation on the relative strength of fluorescence and excitationimages, for example, the relative attenuation of the filters used influorescence measurements, compared to the filters used in excitationlight measurements (see FIG. 2 below). In another arrangement, a=1 andb=1. However, depending upon the application, a and b can be othervalues given by pre-determined equations (for example functions ofwavelength) or they can be experimentally measured.

Similarly a corresponding normalized fluorescence transilluminationimage, U_(t), can be computed as:

$\begin{matrix}{U_{t} = \frac{{a \cdot {\sum\limits_{k = 1}^{Ns}{{g(k)}\left( {{I_{f\; t}(k)} - I_{fn}} \right)_{> {Tf}}}}} + c_{f}}{{b \cdot {\sum\limits_{k = 1}^{Ns}{{g(k)}\left( {{I_{et}(k)} - I_{en}} \right)_{> {Te}}}}} + c_{e}}} & \left( {{Eq}.\mspace{14mu} 4} \right)\end{matrix}$

where I_(et) is a transillumination image obtained at the excitationwavelength (i.e., an intrinsic transillumination image, which is animage of the tissue generated from excitation light generated by atransillumination light source having passed through the tissue) andI_(en) is camera noise (dark image) obtained with no excitation from theexcitation light source. Fluorescence and excitation transilluminationimages for each of the N, sources employed are summed (superimposed)after subtraction with the noise images I_(fn) and I_(en) andapplication of the T_(f) and T_(e) thresholds, respectively.

While noises I_(fn) and I_(en) are subtracted in the above equations 3and 4, it should be understood that the similar normalization techniquesapply to a system in which one or both of the noises are not subtracted.

It should be noted that, for the system 50 described in conjunction withFIG. 1B, which has only the one transillumination light source 52, asingle transillumination image is obtained. In this case Eq. 2 and Eq. 4are written without the summation terms.

An un-normalized fluorescence transillumination image (onetransillumination light source) can be expressed as:

I _(t)=(I _(ft) −I _(fn))_(>Tf)  (Eq. 5)

A normalized fluorescence transillumination image (one transilluminationlight source) can be expressed as:

$\begin{matrix}{{U_{t} = \frac{{a \cdot \left( {I_{f\; t} - I_{fn}} \right)_{> {Tf}}} + c_{f}}{{b \cdot \left( {I_{et} - I_{en}} \right)_{> {Te}}} + c_{e}}},} & \left( {{Eq}.\mspace{14mu} 6} \right)\end{matrix}$

where I_(ft) and I_(et) are now single transillumination images.

The intrinsic image can be an image that is obtained at a broaderwavelength, i.e. at wavelengths that not only include the wavelength ofthe excitation light (e.g., 14, 18 of FIG. 1) but also the wavelength ofresulting fluorescent light. Alternatively two “intrinsic” images can beobtained, one at the wavelength of the excitation light and one at thewavelength of the emitted (fluorescence) light, wherein a combination ofthe two can be used for normalization, for example, a linear combinationor a weighted product.

In the above-described case of intrinsic image generation in which theintrinsic image is obtained at the broader wavelength range thatincludes the fluorescence wavelength, the fluorescence image can firstbe subtracted from the intrinsic image obtained, i.e. an image generatedusing an excitation light having a spectral response that is similar tothe excitation and emission spectral responses, i.e.I_(ec)=I_(ec)′−I_(fe) and I_(et)=I_(et)′−I_(ft), where, as describedabove, I_(ee) is an epi-illumination image obtained at the wavelengthrange of the extended excitation light source, I_(et) is atransillumination image obtained at the wavelength range of the extendedexcitation light source, I_(fe) is the fluorescence image generated whenusing an epi-illumination light source, and, I_(ft) is the fluorescenceimage generated when using a transillumination light source. In thiscase, I_(ee)′ and I_(et)′ are intrinsic images over multiple wavelengthscorresponding to the excitation and emission wavelengths obtained fromepi-illumination and from transillumination, respectively, usingexcitation light having a wavelength at the excitation wavelength andalso additional excitation light having a light spectrum that matchesthe wavelengths of emitted (fluorescence) light. The excitation lightmatching the fluorescence spectrum can be used to capture fluorescencepropagation characteristics. This approach becomes particularlyimportant for cases where the excitation light is in the visiblewavelength range and the fluorescent light is in the near-infraredwavelength range. While the above is described generically, significantaccuracy can be achieved by appropriately selecting the range andresponse over which the I_(ee)′ and I_(et)′ are obtained.

For bioluminescence imaging, Eq. 1, Eq. 3, and Eq. 5 (un-normalizedepi-illumination, normalized epi-illumination, and un-normalizedfluorescence transillumination images, respectively) can remainsubstantially unchanged, but I_(fx) is interpreted to be associated withbioluminescence rather than with fluorescence. In normalizedbioluminescence imaging, images can be normalized with intrinsic imagesobtained at a selected propagation wavelength range that best matchesthe wavelength of the bioluminescence systems. Therefore, excitationlight can also include light having a spectrum that matches thewavelengths of the bioluminescence. Eq. 3 provides a normalizedbioluminescence epi-illumination image.

For bioluminescence imaging, Eq. 2, used for multiple un-normalizedfluorescence transillumination images, is not used. Also, for normalizedtransillumination bioluminescence imaging, Eq. 4, which applies tomultiple transillumination light sources, can be instead expressed as:

$\begin{matrix}{U_{t} = \frac{{a \cdot \left( {I_{f\; t} - I_{fn}} \right)_{> {Tf}}} + c_{f}}{{b \cdot {\sum\limits_{k = 1}^{Ns}{{g(k)}\left( {{I_{et}(k)} - I_{en}} \right)_{> {Te}}}}} + c_{e}}} & \left( {{Eq}.\mspace{14mu} 7} \right)\end{matrix}$

and Eq. 6 for normalized bioluminescence transillumination imaging usingone transillumination light source becomes:

$\begin{matrix}{U_{t} = {\frac{{a \cdot \left( {I_{f\; t} - I_{fn}} \right)_{> {Tf}}} + c_{f}}{{b \cdot \left( {I_{et} - I_{en}} \right)_{> {Te}}} + c_{e}}.}} & \left( {{Eq}.\mspace{14mu} 8} \right)\end{matrix}$

The modification in Eq. 7 (compared with Eq. 4) shows a non-dependenceof the bioluminescence signal on the excitation light sources,therefore, the summation as a function of light sources is dropped.Similarly it should be pointed out that Eq. 7 and Eq. 8 are more genericin nature as they do not explicitly direct the geometry by whichbioluminescence images are captured. However they assume that excitationlight propagates from similar tissues as from which the bioluminescencesignal is collected. One embodiment for normalized imaging given by Eq.3 applied to bioluminescence of superficial structures, whereinbioluminescence images are corrected for tissue attenuation inepi-illumination mode.

It should be appreciated that the normalization methods described aboveare generic in nature and can be applied in both systems 10, 50 of FIG.1 and FIG. 1B (and also systems shown in FIGS. 9, 9A, and 10).

An alternative normalization approach referred to herein as a“per-source” normalization approach, which independently normalizes eachsource that is time, frequency, or wavelength encoded and then adds thenormalized results together. This normalization can be implemented withany light source geometry, even if the light sources are placed on thesides or the top-side of the tissue being imaged.

In accordance with Eq. 4 and the above description, a per-sourcenormalization image can be expressed as:

$\begin{matrix}{U_{psn} = {\sum\limits_{k = 1}^{N_{s}}\frac{{a \cdot \left( {{I_{f\; t}(k)} - I_{fn}} \right)_{> {Tf}}} + c_{f}}{{b \cdot \left( {{I_{et}(k)} - I_{en}} \right)_{> {Te}}} + c_{e}}}} & \left( {{Eq}.\mspace{14mu} 9} \right)\end{matrix}$

The thresholds T_(f) and T_(e) can be constant for all values k or canbe T_(f)=T_(f)(k) and T_(e)=T_(e)(k), which indicates that each ratio,applied for each individual light source, is threshold differently. Inone embodiment, T_(e) is an adaptive threshold that is scaled as afunction of I_(et)(k) intensity, for example T_(e)=0.1*I_(et)(k) andT_(f) is a constant, for example, 5 times the standard deviation ofnoise. Image pixels that give denominator values that are less than T,are not calculated into the summation.

For all equations 1-9, a normalized image may be further processed fordisplay by logarithmically or exponentially relating a final normalizedimage to the above-calculated normalized image. A normalized image fordisplay is selected depending on the dynamic grange of the values in theabove-calculated normalized image.

Subtraction of images in equations above can comprise subtracting pixelmagnitudes of a dark image (noise image) from co-registered pixels of anun-normalized emitted light image (fluorescence or bioluminescence,epi-illumination or transillumination). Division of images in equationsabove can comprise dividing pixel magnitudes of an un-normalized emittedlight image (fluorescence or bioluminescence, epi-illumination ortransillumination) by magnitudes of co-registered pixels of an intrinsicexcitation light image (epi-illumination or transillumination).

While it is described above that the epi-illumination light source 56and the transillumination light source 52 are adapted to generateexcitation light, in other arrangements, the epi-illumination lightsource 56 and/or the transillumination light source 52 are adapted togenerate incident light having the wavelength of the excitation lightand also other wavelengths. The same is true for other arrangements, forexample, arrangements shown in FIGS. 9, 10, and 11, which are describedmore fully below.

Referring now to FIG. 1C, a normalization processor 80, which may be thesame as or similar to the normalization processors 30, 70 of FIGS. 1 and1B, respectively, receives one or more intrinsic excitation light imageson path 82 and one or more un-normalized emitted light images on path 83and generates one or more of a variety of types of a normalized lightimages made available on signal paths 93-96.

The intrinsic excitation light images on path 82 are received by anoptional intrinsic image combining processor 84. The intrinsic imagecombining processor 84 can be adapted to combine (e.g. superimpose)intrinsic transillumination images for systems having a plurality oftransillumination light sources, e.g., die system 10 of FIG. 1, and togenerate a single intrinsic image at an output thereof on path 84 a. Inother arrangements, the intrinsic image combining processor 84 isadapted to combine intrinsic images generated at a plurality ofwavelengths. For a system having only one transillumination lightsource, for example, the system 50 of FIG. 1B, or a system for which theexcitation light source generates light at only the excitation lightwavelength, the intrinsic image combining processor 84 is not needed.

Similarly, the un-normalized emitted light images on path 83 arereceived by an optional emitted image combining processor 85. Theemitted image combining processor 85 is adapted to combine (e.g.superimpose) emitted transillumination images for systems having aplurality of transillumination light sources, e.g., the system 10 ofFIG. 1, and to generate a single un-normalized emitted light image at anoutput thereof on path 85 a. For a system having only onetransillumination light source, for example, the system 50 of FIG. 1B,the emitted image combining processor 85 is not needed.

An intrinsic image noise reduction processor 86 is adapted to receivethe single intrinsic image on path 84 a and also a dark image on path 82a (background image). The intrinsic image noise reduction processor 86is further adapted to combine the single intrinsic image and the darkimage to provide a noise-reduced intrinsic image-on path 86 a. In someembodiments in which noise reduction is not performed, the intrinsicimage noise reduction processor 86 is not used and the single intrinsicimage on path 84 a is used in place of the noise-reduced intrinsic imageon path 86 a.

Similarly, an emitted image noise reduction processor 87 is adapted toreceive the single un-normalized emitted light image on path 85 a andalso a dark image on path 83 a (background image). The emitted imagenoise reduction processor 87 is further adapted to combine the singleun-normalized emitted light image on path 85 a and the dark image onpath 83 a to provide a noise-reduced un-normalized emitted light imageon path 87 a. In some embodiments in which noise reduction is notperformed, the emitted image noise reduction processor 87 is not usedand the un-normalized emitted light image on path 85 a is used in placeof the noise-reduced un-normalized emitted light image on path 87 a.

When the noise-reduced intrinsic image on path 86 a is a noise-reducedintrinsic epi-illumination image and when the noise-reducedun-normalized emitted light image on path 87 a is a noise-reducedun-normalized fluorescence epi-illumination image, those images areprovided to a fluorescence epi-illumination normalization processor 89adapted to generate a normalized fluorescence epi-illumination image onpath 93.

When the noise-reduced intrinsic image on path 86 a is a noise-reducedintrinsic transillumination image and when the noise-reducedun-normalized emitted light image on path 87 a is a noise-reducedun-normalized fluorescence transillumination image, those images areprovided to a fluorescence transillumination normalization processor 90adapted to generate a normalized fluorescence transillumination image onpath 94.

When the noise-reduced intrinsic image on path 86 a is a noise-reducedintrinsic transillumination image and when the noise-reducedun-normalized emitted light image on path 87 a is a noise-reducedun-normalized bioluminescence transillumination image, those images areprovided to a bioluminescence transillumination normalization processor91 adapted to generate a normalized bioluminescence transilluminationimage on path 95.

When the noise-reduced intrinsic image on path 86 a is a noise-reducedintrinsic epi-illumination image and when the noise-reducedun-normalized emitted light image on path 87 a is a noise-reducedun-normalized bioluminescence epi-illumination image, those images areprovided to a bioluminescence epi-illumination normalization processor92 adapted to generate a normalized bioluminescence epi-illuminationimage on path 96.

The normalization processor 80 is adapted to generate the four types ofimages on paths 93-96, wherein each one of the images can be noisereduced or not. Furthermore, the normalized fluorescencetransillumination image provided on path 94 and the normalizedbioluminescence transillumination image provided on path 95 can begenerated using one excitation transillumination light source or aplurality of excitation transillumination light sources (superimposedvia the processors 84, 85). However, in other arrangements, only one ormore of the processors 89-92 are included in the normalization processor80.

Referring now to FIG. 2, a camera 100, which can be the same as orsimilar to the camera 20 of FIG. 1 and the camera 60 of FIG. 1B, isadapted to receive and process light.

The light received by the camera 100 can include intrinsic excitationlight resulting from excitation light generated by an epi-illuminationlight source, and/or fluorescent light emitted by a tissue in responseto the excitation light generated by the epi-illumination light source.The light received by the camera 100 can also include excitation lightresulting from excitation light generated by a transillumination lightsource, and/or fluorescent light emitted by a tissue in response to thetransillumination light generated by the transillumination light source.The light received by the camera 100 can also include bioluminescencelight naturally generated within the tissue.

The camera 100 can include an intrinsic image processor 102, which isadapted to receive the above-described excitation light resulting fromeither the epi-illumination light source or the transillumination lightsource that has impinged upon and interacted with the tissue, and whichis also adapted to generate an intrinsic image (i.e., intrinsic imagedata).

The camera 100 can also include a fluorescence image processor 104,which is adapted to receive the above-described fluorescent lightresulting from either the epi-illumination light source or thetransillumination light source that has been emitted from the tissue,and which is also adapted to generate an un-normalized fluorescenceimage (i.e., un-normalized fluorescence images data). The intrinsicimage processor 102 and the fluorescence image processor 104 can beprovided, for example, by way of or in conjunction with an opticalfilter (excitation light filter 106) adapted to pass the excitationlight and another optical filer (fluorescent light filter 108) adaptedto pass the fluorescent light. The intrinsic image processor 104 and thefluorescence image processor 106 can operate at substantially the sametime or in sequence to provide the intrinsic image (i.e., intrinsicimage data) and the un-normalized fluorescence image (i.e.,un-normalized fluorescence image data). Since the excitation lightreceived by the camera 100 may be at a greater intensity than thefluorescent light, in some embodiments, the excitation light filter 106can include a light attenuating filter, for example, a neutral densityfilter.

In some embodiments, polarizers (not shown) can be further used topreferentially select polarization modes. The polarizers can be used atthe epi-illumination light source (e.g. 56, FIG. 1B), at thetransillumination light source (e.g., 52, FIG. 1B), at the intrinsicportion 102 of the camera 100, and/or at the fluorescence processor 104of the camera 100. The polarizers can facilitate more superficialimaging (when the same orientation polarization filters are used at eachend of a light path) or deeper imaging (when cross-polarizers are used).In addition, polarizers can be used to better separate emission(fluorescence or bioluminescence) from excitation light by polarizingthe emission light with one polarization direction and detecting emittedlight in with a cross polarization.

While the fluorescence image processor 104 is described above, it shouldbe understood that the same processor can be used to receive lightresulting from bioluminescence and to form a bioluminescence image.While the excitation light filter 106 is described above, in otherarrangements, the excitation light filter 106 is replaced with anincident light filter adapted to pass a plurality of wavelengths.

Referring now to FIG. 2A, another camera 150, which can be the same asor similar to the camera 20 of FIG. 1 and the camera 60 of FIG. 1B, isadapted to receive and process light. The camera 150 can include afilter wheel assembly 152 adapted to rotate and to select either anexcitation light filter 154 or a fluorescent light filter 154. An imageprocessor 158 is adapted to generate an intrinsic image or anun-normalized fluorescence image accordingly.

Unlike the camera 100 of FIG. 2, the camera 150 has only one imageprocessor 158, which is used to sequentially provide both the intrinsicimage (i.e., intrinsic image data) and the un-normalized fluorescenceimage (i.e. un-normalized fluorescence image data). Since the excitationlight received by the camera 100 may be at a greater intensity than thefluorescent light, in some embodiments, the excitation light filter 154can include a light attenuating filter, for example, a neutral densityfilter. While the excitation light filter 154 is described above, inother arrangements, the excitation light filter 154 is replaced with anincident light filter adapted to pass a plurality of wavelengths.

Before describing a series of images presented below, phantoms used togenerate the images are described. A first phantom (used in images ofFIGS., 3, 3A, and 4) was relatively spatially-homogenous and includedtwo diffusive and fluorescent tubes placed 5 mm apart, which wereimmersed in a chamber. The tubes were glass capillaries ˜1.5 mm diametersealed on one end, containing 1% intralipid solution, 25 ppm of Indiaink, and 200 nm of Cy 5.5 dye to simulate varying absorption andfluorescence emanating from a superficial tumor. The chamber was furtherfilled with the same solution of intralipid and ink but without thefluorochrome. The first phantom was placed at various depths in thechamber and was used to assess the relative performance of the abovedescribed normalization method as a function of phantom depth.

A second phantom (used in images of FIGS. 5-5C) employed the samerelative homogenous background and a two-tube arrangement as the firstphantom but employed two 3 mm diameter plastic tubes. The second phantomwas used to examine accuracy of the two normalization methods as afunction of varying optical properties. In these experiments the lefttube was filled with 400 nm of Cy5.5 whereas the right tube contained200 nm of Cy5.5. Both tubes and the background medium were filled withthe same solution of 1% intralipid solution and 25 ppm of India ink(FIGS. 5 and 5B). Subsequently the absorption of the left tube wasincreased to 2.5× the background absorption concentration (FIGS. 5A and5C). The second phantom was used to examine robustness of the normalizedimaging methods with respect to varying background optical properties.

A third phantom was used in a first animal experiment (shown in FIGS. 6and 6A) to generate images of a nude mouse post-mortem. This experimentdemonstrated the merits of normalized transillumination in resolvingdeep-seated activity, better than epi-illumination. It also demonstratedhow normalized epi-illumination can result in reducing false positives.The third phantom included a 1.8 mm diameter glass tube filled withintralipid and 400 nm of Cy5.5 dye, which was inserted in the animalthrough the esophagus until it reached the middle of the torso of theanimal. Then the animal was placed in the imaging chamber and all imagesets described by Eqs. 1-4 were obtained.

Images shown in FIG. 6A and FIG. 7A are negative images. However,positive images could equally well be generated and shown. It will alsobe understood that the intrinsic, un-normalized epi-illuminationfluorescence, un-normalized transillumination fluorescence, normalizedepi-illumination fluorescence, and normalized transilluminationfluorescence images presented in FIGS. 6, 6A, 7, and 7A are allprocessed to remove background noise, for example, as in equations 1-4above.

Referring now to FIG. 3, normalized fluorescence epi-illumination imagesof the first phantom are shown in panels (a)-(e), where theabove-described tubes are placed at different depths in the chamber asindicated at 0, 1, 3, 5 and 7 mm away from a front glass window of thechamber. At a depth of 0 mm the tubes are physically in contact with thefront window. The images in panels (a)-(e) are scaled to their maximum,since signal intensity drops exponentially with depth.

In this particular experiment, fluorescence epi-illumination imagingquality appears reduced as a function of depth. The tubes of the firstphantom are practically not detectable at a depth of 7 mm. However,normalized fluorescence epi-illumination imaging yields significantresolution of the tubes at 0 mm. The two tubes are clearly resolved at 0mm (when they are in contact with the front window). In contrast, itwill be shown below, that at 0 mm, the tubes are less well resolved innormalized fluorescence transillumination imaging.

Referring now to FIG. 3A, normalized fluorescence transilluminationimages of the first phantom are shown in panels (a)-(e). Again, theimages in panels (a)-(e) are scaled to their maximum since signalintensity drops exponentially with depth.

The tubes are detected in all normalized transillumination images at avariety of depths, although resolution appears to deteriorate as afunction of depth. Broadening of the detected image of the tubes is seenin the fluorescence transillumination images as a function of depth asexpected. Moreover, as the arrows indicate, there is a length dependentbroadening as well. However the ability of normalized transilluminationto detect the tubes deeper than normalized epi-illumination (FIG. 3) isapparent and may be important for improving planar images of sub-surfaceactivity and deep-seated activity.

Referring now to FIG. 4, transillumination images of the first phantomare shown for the tubes placed at a depth of 5 mm. In panel (a), anintrinsic image of excitation light is generated. As described above,the excitation light corresponds to light from a transillumination lightsource having passed through tissue, here through the chamber and firstphantom. The region of excitation light measures ˜3×2 cm and itsintensity exponentially drops outside this area.

In panel (b), an un-normalized fluorescence transillumination image iscollected. As described above, the fluorescent light is emitted by afluorescent probe within the tubes in response to the emitted light fromthe transillumination light source. The image of panel (b) is anun-normalized image, I_(t), according to equation 2.

In panel (c), a normalized fluorescence transillumination image, U_(t),is shown according to equation 4. The image, U_(t), more accuratelyrepresents a true length of the tubes than the image, It, of panel (b).This is because the tubes are not covered homogeneously by theexcitation light of panel (a). It would be advantageous to homogeneouslyilluminate the complete region of interest, however, as shown, thenormalization can accurately represent dimensions of the tubes even wheninhomogeneous excitation light is used. However, due to an asymmetry ofthe excitation light field of panel (a), there is an asymmetry ofresolved “shape” at the ends of the image of the tubes (panel (c)).

Normalized fluorescence transillumination imaging is seen to be lesssensitive to illumination field variations and also less sensitive todepth than normalized fluorescence epi-illumination imaging and thanun-normalized fluorescence transillumination imaging. However, as shownby comparing FIG. 3, panel (a), with FIG. 3A, panel (a), normalizedfluorescence epi-illumination imaging can be seen to provide higherresolution images than normalized fluorescence transillumination imagingfor objects near a surface of a diffuse medium. This advantage isreduced even at a fluorescent probe depth of 1 mm below the surface.

Further experimental results described below show both normalizedfluorescence transillumination images and normalized fluorescenceepi-illumination images.

Referring now to FIGS. 5-5C, images were obtained with fluorescenceepi-illumination and fluorescence transillumination imaging of thesecond phantom described above. As described above, the second phantomincludes two tubes immersed in a solution of 1% intralipid solution and25 ppm of India ink. In FIGS. 5 and 5B the tubes contained 25 ppm ofIndia ink, i.e. the same as the background concentration. In FIGS. 5Aand 5C the left tube contained 60 ppm India ink, i.e. ˜2.5× thebackground ink concentration.

Referring first to FIGS. 5 and 5A, panels show intrinsic (excitation)light images (panels (a)), uni-normalized fluorescence epi-illuminationimages (panels (b)), and normalized fluorescence epi-illumination images(panels (c)), respectively. By comparing the un-normalized fluorescenceepi-illumination images (panels (b)) with the normalized fluorescenceepi-illumination images (panels (c)), the effect of added absorption ofthe left tube can be seen. At 2.5× concentration (FIG. 5A) theun-normalized fluorescence image (panel b)) show only a 1.26:1 intensityratio over the right tube where the actual ratio is 2:1 as shown at 1×concentration (panel b, FIG. 5). However, the normalized fluorescenceimages (panels (c)) show an improved ratio of 1.41:1.

Referring now to FIGS. 5B and 5C, images are of the same phantom as inFIGS. 5 and 5A including the above-described 1× or 2.5× inkconcentration contained in the left tube as indicated. Panels showtransillumination intrinsic images (panels (a)), un-normalizedfluorescence transillumination images (panels (b)), and normalizedfluorescence transillumination images (panels (c)), respectively.

Here, the un-normalized fluorescence transillumination images (panels(b)) show a 1.11:1 ratio, which is worse than the epi-illuminationun-normalized fluorescence images (panels (b)) of FIGS. 5 and 5A.However, the normalized fluorescence transillumination images (panels(c)) show a ratio of 1.58:1, which is the better than the normalizedfluorescence epi-illumination images (panels (c)) of FIGS. 5 and 5A.Therefore, normalized fluorescence transillumination imaging is shown toaccurately measure fluorescence activity, here of superficial volumes,when the background optical properties are heterogeneous, as is the casewhen imaging most tumors and other diseases and medical conditions.

Referring now to FIG. 6, panels (a)-(c) show an epi-illuminationintrinsic image, an un-normalized fluorescence epi-illumination image,and a normalized fluorescence epi-illumination image, respectively, of anude mouse implanted with a fluorescent tube post-mortem. Panel (a) alsoshows an approximate position of the tube (see dark arrows) in theviewing plane as indicated by a dashed outline (repeated in panels (b)and (c)), found by measuring the insertion distance and bypost-experiment surgical removal. The normalized fluorescenceepi-illumination image of panel (c) does not show the implanted tube. Intact, the normalized image of panel (c) has less contrast than theun-normalized image of panel (b).

A white arrow in panel (b) shows fluorescence where no fluorescencesignal should be present in the un-normalized fluorescence image. Thisregion of fluorescence is reduced in the normalized image of panel (c),as is also shown by a white arrow in panel (c). This reduction showsthat the normalization method can reduce false positives.

Skin auto-fluorescence and bleed through signals (e.g., reception ofdirect light along paths from the light source to the camera) appear instandard un-normalized fluorescence epi-illumination images, andnormalization provides a more uniform epi-illumination image with fewerartifacts. Therefore, a very low or no signal image in fluorescence isexpected, since there is very low auto-fluorescence in the nearinfrared. This is shown by the low contrast normalized image (panel(c)).

Another advantage of the normalized fluorescence epi-illumination imageis that it can better identify an absence of fluorescence. This is animportant feature for intra-operative imaging, laparoscopic imaging,endoscopy, and small animal imaging, since it accounts for variationsand reflections of the excitation light that may bleed through to thecamera or that may selectively excite more background auto-fluorescence.Therefore normalized fluorescent epi-illumination imaging can offerimproved imaging accuracy and performance when used for its negativepredictive capability.

Referring now to FIG. 6A, panels (a)-(c) show an intrinsic image, anun-normalized fluorescent transillumination image, and a normalizedfluorescence transillumination image, respectively, of the animal ofFIG. 6. The tube (at arrows) is detected and it is better resolved inthe normalized fluorescence transillumination image than in thenormalized fluorescence epi-illumination image (panel (c), FIG. 6). Thetube size is overestimated in panel (c) but its two-dimensional locationin the viewing plane is well resolved. Transillumination imaging hasbeen performed with mice in the presence or absence of matching fluidswith very similar results.

The images of FIGS. 6 and 6A show that normalized transilluminationimaging, and similarly normalized epi-illumination imaging, can operatein the absence of matching fluids. However, proper care should be takento selectively attenuate light at the borders of the tissue and outsidethe borders of the tissue to yield optical matching to the dynamic rangeof the measurements to the light detector used.

Referring now to FIG. 7, panels (a)-(c) show an intrinsicepi-illumination image, a un-normalized fluorescent epi-illuminationimage, and a normalized fluorescence epi-illumination image,respectively, of an animal bearing a surface tumor with an ellipsoidshape of 4 mm×3 mm long and short axis dimensions, respectively, asdetermined by caliper measurements. The tumor is an Erb2 positivespontaneous tumor in the left mammary fiat pad (right side as shown onthe image). The animal was injected with a cathepsin-sensitivefluorescent probe. The tumor is indicated by an arrow on the images.This tumor was highly vascular and highly absorbing and was thereforeseen darker than surrounding tissue on the intrinsic epi-illuminationimage shown in panel (a). A corresponding un-normalized fluorescenceepi-illumination image shown in panel (b) does not identify the tumor.The normalized fluorescence epi-illumination image shown in panel (c)provides an image that appears more flat (i.e., the same intensitythroughout the animal) and the tumor is now identifiable. However thedetection ability is reduced due to other fluorescence activity withinthe animal, i.e., outside of the tumor, resulting from fluorochromeoutside of the tumor.

Referring now to FIG. 7A, panels (a)-(c) show an intrinsictransillumination image, an un-normalized fluorescence transilluminationimage, and a normalized fluorescence transillumination image,respectively, of the animal of FIG. 7. The normalized image of panel (c)shows a marked fluorescence increase associated with the tumor (arrow).The tumor is not visible in panel (b), the un-normalized fluorescencetransillumination image.

The animal of FIGS. 7 and 7A was injected with a fluorescence probe,therefore, it is expected that there is a remnant backgroundfluorescence signal in tissues apart from the tumor. This higherbackground image signal is evident in panel (c) (compare to panel (c) ofFIGS. 6A and 6B). However the detection improvement gained by thenormalized fluorescence transillumination method is still evident.

The above system and methods can also apply to bioluminescence imaging,wherein an un-normalized bioluminescence image is normalized with anintrinsic epi-illumination image to yield a normalized bioluminescenceepi-illumination image. While tissue emission apart from a tumor is lessof an issue for bioluminescence than for fluorescence(auto-fluorescence), the normalization can nevertheless correct forattenuation heterogeneity across the animal surface, which can resultfrom blood concentration heterogeneity, especially if a light sourcewith spectral characteristics to match those of the emittedbioluminescence is used. Similarly the bioluminescence image can benormalized with an intrinsic transillumination image, and this mayachieve a better result that than normalizing with an intrinsicepi-illumination image, especially when the bioluminescence is emittedfrom lesions deeper in the animal.

Referring now to FIG. 8, images show another mouse having an Erb2positive spontaneous tumor, which was imaged after the administration ofa cathepsin sensitive activatable fluorescence probe. These imagesdemonstrate improved images obtained with normalized epi-illuminationimaging, which allow improved identification of the location of afluorescent lesion (at arrows). It can be seen that normalizedepi-illumination imaging can be useful for lesion identification whenused in surgical operations, endoscopy, and laparoscopy. Here normalizedimages both before (panel (c)) and after (panel (d)) noise removal areshown.

Referring now to FIG. 8A, images show a mouse having two HT1080 tumorsimplanted in the left and right side of the mammary fat pads, which areimaged after the administration of a cathepsin sensitive activatablefluorescence probe. These images demonstrate improved images obtainedwith normalized epi-illumination imaging, which allow improvedidentification of tumor borders. It can be seen that normalizedepi-illumination imaging can be useful for border identification whenused in surgical operations, endoscopy, and laparoscopy. Images bothbefore (panel (c)) and after (panel (d)) noise removal are shown.

FIGS. 8 and 8A show images related to techniques, which can be used inmedical procedures, for example, intra-operative imaging, endoscopy, orlaparoscopy used for the identification of lesions and their borders.

Referring again to FIG. 8, panels (a)-(d) show an epi-illuminationintrinsic image, an un-normalized epi-illumination fluorescent lightimage, a normalized fluorescence epi-illumination image that assumesnoise thresholds at zero, and a normalized fluorescence epi-illuminationimage that has been processed with noise threshold operations asdescribed in Eq. 3, respectively. The images show an animal bearing asurface tumor, as indicated with an arrow, which appears dark on theintrinsic epi-illumination image (panel (a)). Due to tumor absorption oflight, the tumor appears dark on the un-normalized fluorescenceepi-illumination image (panel (b)). Some activity that can be observedat the top part of the tumor is not co-localized with the presence ofthe tumor, even if the animal has been injected with the sameactivatable fluorescent probe as in FIG. 7. Such activity at the bordersof the tumor is typical in some un-normalized images and may be due to alocal excitation field variation resulting from tissue heterogeneousabsorption. Conversely the normalized fluorescence epi-illuminationimages of panels (c) and (d) correctly identify the location and size ofthe tumor. The image of panel (d) has been enhanced by the applicationof noise thresholds and by maximizing contrast. An additional smallerlesion also appears on the left side of the large tumor and this isvisualized well in both normalized fluorescence images. However, inparticular, the image of panel (d) demonstrates how the normalizedfluorescence image can better show the total background fluorescenceactivity.

Referring again to FIG. 8A, images show the same configuration as shownin FIG. 8. A mouse has two HT1080 implanted tumors, which are indicatedby arrows. Borders of the right tumor are not well differentiated sincelight “leaks” into the low absorbing skin fold that forms just above it.Therefore, the tumor appears as elongated. This characteristic may bedrawback in surgical procedures where the exact tumor margins need to beidentified so that unnecessary tissue damage during operation isminimized, especially, for example, in brain surgery. Conversely thenormalized images of panels (c) and (d) better differentiate the tumormargins, which was also was correlated by actual tumor excision andinvasive inspection.

Referring now to FIG. 9 a system 200 for intra-operative (i.e., imagingduring an operation) procedures includes a camera 202, which can includetwo lenses and/or filters 204 a, 204 b. The camera 202 with filters 204a, 204 b can be the same as or similar to the camera 100 (includingfilters 106, 108) of FIG. 2. The system 200 can also include anepi-illumination light source 206, which can, in one particularembodiment, be in the form of a ring illuminator. The camera can provideun-normalized images to a normalization processor 208, which can providenormalized images to a monitor 210.

It will be appreciated that the system 200 is similar to the system 10of FIG. 1, however, the system 200 can be reduced in size so as toprovide a system that can be more easily used during an operation.

In use, a body part or organ 212 can be exposed as shown, for example,by a surgical incision. A fluorochrome, which can be injected into thepatient before or during the operation, and which tends to congregate atthe site of a lesion 216, makes the lesion fluorescently reactive. Asdescribed above, the camera 202 can generate an intrinsicepi-illumination image and an un-normalized fluorescenceepi-illumination image. The normalization processor 208 can generate anormalized epi-illumination fluorescence image and can send thenormalized image to an intra-operative monitor 210, where it can beviewed by a surgeon.

In one particular embodiment, the intra-operative monitor 210 is inproximity to the operation theater so that the surgeon can seefluorescence indicative of disease on the monitor.

Also shown in FIG. 9, an illuminator/laparoscope/endoscope 218(hereafter called a probe 218) can be used in a variety of ways. In onearrangement, the probe 218 provides a transillumination light source,which can be used so that the camera 202 can capture an intrinsictransillumination image and/or an un-normalized fluorescencetransillumination image of a lesion 220. This probe can be used duringsurgery to complement the epi-illumination system 200 or it can provideonly transillumination imaging without use of the epi-illumination lightsource.

In another arrangement, the probe 218 provides an epi-illumination lightsource, wherein the system 200 is coupled to a distal end of the probe218 in order to generate both an intrinsic epi-illumination image and anun-normalized fluorescence epi-illumination image and also a normalizedfluorescence epi-illumination image of the lesion 220 in response to theepi-illumination light source provided by the probe 218. Sucharrangements will be better understood form the discussion below inconjunction with FIGS. 11 and 11A.

In some arrangements, the probe 218 can be manually manipulated duringthe operation to image selected structures or organs. Real-timenormalization techniques, such as the one described by Eq. 4, can beimplemented using last image processing digital processors, and canprovide accurate imaging independent of the exact pattern of theillumination field, which may vary when generating differentillumination orientations when manipulating the probe 218.

The probe 218 can be placed, for example, on the side of or behind alymph node to investigate or confirm the presence or absence of afluorochrome indicative of a malignancy. The probe 218 can be placedbehind an organ to identify fluorescence activity indicative of functionor disease. This fluorescence is provided by a local or systemicadministration of a fluorochrome.

In some embodiments, as described above, the probe 218 is an endoscopeor laparoscope so that the fluorescence activity can be captured by theprobe 218. These arrangements can be used, for example, in laparoscopy,minimally invasive surgery, endoscopy, and colonoscopy.

Referring now to FIG. 9A, in another embodiment, the system 200 of FIG.9 is constructed entirely or in part within a miniaturized detection andvisualization system, for example, goggles 230 or a transparent screen(not shown). These embodiments can allow, via the use of appropriatefilters, co-registration of anatomical contrast, as would be naturallyseen by the eye, in combination with fluorescence activity. For example,in one arrangement, the body/organ 212 is seen naturally and colorcontrast is added to indicate the presence of the fluorescenceassociated with a lesion.

In yet another embodiment, the system 200 is a portable system, forexample a hand-held scanner.

Referring now to FIG. 10, a system 250 includes a transilluminationlight source 252 disposed on the opposite side of a tooth 254 from acamera 256. The camera 256 can have two filters 258 a, 258 b, and can bethe same as or similar to the camera 100 of FIG. 2. The camera 256 cangenerate an intrinsic transillumination image and also andtin-normalized fluorescence transillumination image of the tooth 254 andstructures therein. In some arrangements the camera is instead anendoscope, which can be the same as or similar to the probe of FIG. 9. Anormalization processor (not shown) and monitor (not shown) can becoupled to the camera 256 in order to generate normalized images.

In this embodiment, the transillumination light source 252 and thecamera 256 are introduced in the mouth and disposed about the tooth 254as shown. In some arrangements, the camera 256 also includes anepi-illumination light source (not shown), which can be the same as orsimilar to the epi-illumination light source 206 of FIG. 9.

The system 250 can generate normalized fluorescence transilluminationimages and/or normalized fluorescence epi-illumination images of dentalfluorescence contrast. In some embodiments, the system 250 can usenative tissue auto-fluorescence, which can be excited by the excitationlight source 252. However, in other embodiments, extrinsically ortopically administered external fluorochromes with molecular orfunctional specificity can be applied to the tooth 254.

The system 250 can also be used to investigate other oral cavitydiseases, for example gum disease or oral cancers. The system 250 can beused in combination with other non-invasive imaging systems, for exampleX-ray systems, to provide further diagnostic capability.

Referring now to FIG. 11, a system 270, used for colposcopic proceduresincludes a colposcopic probe 272 (or more simply a probe 272) having aface 272 a. The face 272 a of the probe 272 is described in greaterdetail below in conjunction with FIG. 11A. The probe 272 is insertedinto the colon 274 of a patient and is coupled with a colposcope 276 toa colposcope coupler 278. The colposcope 276 includes at least one lightfiber (not shown) therewithin.

The colposcope coupler 276 is optically coupled to a light coupler 280,which is adapted to receive light from at least one excitation lightsource (not shown) and to deliver the light to the colposcope 276. Here,two illumination lights are shown, which are represented by arrows 282,284. In some embodiments, illumination channel 1 282 providesepi-illumination excitation light at an excitation light wavelength,which is associated with normalization system and method of the presentinvention. In some embodiments, illumination channel 2 284 also providesepi-illumination excitation light at the same excitation lightwavelength. However, in some embodiments, the illumination channel 2284, can provide light at another wavelength, for example, visible lightwavelengths, and is associated with other optical functions, forexample, direct colposcopic viewing of visible light as will be apparentfrom discussion below.

In further embodiments, the epi-illumination channel 2 provides anotherexcitation epi-illumination light at another excitation lightwavelength. In these embodiments, the first illumination channel 1 282is used to excite certain fluorochromes within the colon 274 and thesecond illumination channel 284 is used to excite other fluorochromeswithin the colon 274. With this arrangement, two different normalizedfluorescence images can be generated.

The colposcope coupler 276 is also optically coupled to a light splitter288, which is adapted to pass a first portion 288 of received light to acamera/monitor 290. The camera/monitor 280 can be used, for example, fordirect colposcopic viewing of visible light during a procedure. A secondportion 291 of the received passes through a light filter 292, which iscoupled to an optical coupler 294, which directs the second receivedlight 291 to a camera 298. The camera 298 can be the same as or similarto the camera 100 of FIG. 2 or the camera 150 of FIG. 2A. From the abovediscussion, it should be appreciated that the first and second portions288, 291, respectively, of the received light can include excitationepi-illumination light at an excitation light wavelength, fluorescencelight a fluorescence wavelength, epi-illumination light at anotherexcitation light wavelength, fluorescent light at another fluorescencewavelength, and visible light, in any combination.

The camera 298 is shown to be most similar to the camera 100 of FIG. 1,and has an intrinsic image processor 300 and a fluorescence imageprocessor 302. With this arrangement, the filter 292 can be the same asor similar to the excitation light filter 106 and the fluorescent lightfilter 108 of FIG. 2.

The camera provides intrinsic excitation epi-illumination images 304 andun-normalized fluorescence epi-illumination images 304 to anormalization processor 306. The normalization processor 306 can be thesame as or similar to the normalization processors 30, 70, 80 of FIGS.1, 1B, and 1C, respectively. The normalization processor 306 is adaptedto generate normalized fluorescence epi-illumination images 308 to amonitor 310.

The system 270 is but one example of a system that can be usedintra-operatively to provide normalized fluorescence epi-illuminationimages. Other similar systems can be used to provide normalizedfluorescence transillumination images, as described above in conjunctionwith FIG. 9.

A system similar to the system 270 can be used in a variety of operativeprocedures, including, but not limited to, a laparoscopic procedure(wherein the probe 272 is a laparoscopic probe), an endoscopic procedure(wherein the probe 272 is an endoscopic probe), a colonoscopic procedure(wherein the probe 272 is a colonoscopic probe), a colposcopic procedure(wherein the probe 272 is a colposcopic probe), an esophageal procedure(wherein the probe 272 is an esophageal probe), a pulmonary procedure(wherein the probe 272 is a pulmonary probe), an oral procedure (whereinthe probe 272 is an oral probe), and a dental procedure (wherein theprobe 272 is a dental probe). A system similar to the system 270 can beused to image any internal body cavity within a patient.

Referring now to FIG. 11A, the face 272 a of the probe 272 (FIG. 11) caninclude a first illumination port 350 from which the illumination light282 emanates, and a second illumination port 352 from which theillumination light 282 emanates. The face 272 a can also include a porthaving a camera lens 354, which provides the portions 288, 291 (FIG. 11)of received light to the camera 298 (FIG. 1). The face 272 a can alsoinclude an irrigation port 356 through which fluid can be pumped intothe colon 274 (FIG. 1) or retrieved from the colon 274. The face3 272 acan also include a channel 358 through which instruments can be insertedinto the colon 274.

The term “optical biopsy” used in conjunction with reference designator358 is used to indicate an optical method, which is used tonon-invasively acquire tissue information similar to the way in whichinformation is obtained by a traditional invasive biopsy (i.e. tissueexcision and histological examination). For example, an optical biopsycan be a spectroscopic technique that locally assesses cell nuclear sizeand density, an in-vivo confocal microscopy technique, or a normalizedfluorescence method such as the one described herein. Similar to allinvasive biopsy, an optical biopsy is used to characterize a relativelysmall lesion. A biopsy is used to characterize a relatively small areawith high specificity in contrast to so-called “screening” that isassociated with large fields of view.

It should be appreciated that all of the ports, and channels of the face272 a are associated with light fibers or channels through thecolposcope 276 of FIG. 1.

It will be appreciated from the image examples provided in FIG. 5-8A,that normalized epi-illumination and normalized fluorescencetransillumination imagining provide improved images in a variety ofapplications. Normalized epi-illumination imaging tends to be surfaceweighted. In other words, surface features can be seen well and withepi-illumination imaging the technique is appropriate for surgical andendoscopic applications. However the sensitivity and imaging resolutionof epi-illumination imaging can be reduced for objects that are underthe surface, in which case, normalized fluorescence transilluminationimaging may be preferred. Normalized fluorescence transilluminationimagining can often provide useful images in diffusive volumes togreater depths than normalized fluorescence epi-illumination imaging.This is because normalized fluorescence transillumination imagining hasdifferent characteristics than normalized fluorescence epi-illuminationimaging. In particular, only a relatively small amount of excitationlight is collected in normalized fluorescence transilluminationimagining due to attenuation of the excitation light as it passesthrough the diffusive volume, therefore, any bleed through signal isreduced. Similarly, in normalized fluorescence transilluminationimagining, any surface auto-fluorescence is excited by significantlyattenuated light compared to normalized fluorescence epi-illuminationimaging. These differences yield more uniform volume sampling intransillumination and significantly reduced background signals.

The above-described normalization can improve imaging performance bycorrecting for the effects of optical property variation in tissues andfor variations in the strength of the excitation illumination field.Normalization is well suited for in-vivo investigations and can beimplemented as a real time correction so that the user can observecorrected images in real time.

In general, it will be appreciated that un-normalized transilluminationimages can serve as raw data for conventional tomographic images. Whenused for improved tomographic imaging, normalized fluorescencetransillumination images can provide improved image volume (i.e.increased depth), minimization of surface fluorescence, and reducedsensitivity to variations of transillumination light source opticalproperties. Due to the use of accurate photon propagation models andsubsequent inversion, tomography using normalized images in place ofun-normalized images can yield improved three-dimensional quantifiedmaps of fluorescence activity and can correct for depth dependentsensitivities of collected photons. Therefore, tomography is a moreintegrated approach than either epi-illumination or transilluminationplanar imaging. Tomography is, however, more complex to implement anduses relatively complex tomographic processing. Tomography also suffersfrom longer computation times, which tend to make tomography lessfavored when compared to epi-illumination and transillumination planarapproaches, especially when used for real time imaging.

It should be also appreciated that imaging performance can besignificantly improved if normalized fluorescence epi-illumination andnormalized fluorescence transillumination images and methods are used incombination. Using a combined normalized epi-illumination/normalizedtransillumination system as shown, for example, in FIGS. 1 and 1B, thesensitivity of epi-illumination imaging to surface activity is retained,but volume sampling (i.e., resolution with depth) capability oftransillumination imaging is added.

In both epi-illumination and transillumination systems and methods,normalization can offer certain advantages, including, but not limitedto, reduced sensitivity to inhomogeneous illumination field variationsand variations in background optical properties. Hardware requirementsare only moderately increased for a combined epi-illumination andtransillumination system.

All references cited herein are hereby incorporated herein by referencein their entirety.

Having described preferred embodiments of the invention, it will nowbecome apparent to one of ordinary skill in the art that otherembodiments incorporating their concepts may be used. It is felttherefore that these embodiments should not be limited to disclosedembodiments, but rather should be limited only by the spirit and scopeof the appended claims.

1. A method of imaging, comprising: generating incident light includingexcitation light with an excitation light source; directing the incidentlight toward a tissue; receiving the incident light with a lightdetector after the incident light has interacted with a tissue;receiving emitted light with the light detector, wherein the emittedlight is emitted from the tissue; generating an intrinsic image of thetissue in response to the incident light; generating an un-normalizedemitted light image of the tissue, and combining the un-normalizedemitted light image and the intrinsic image to generate a normalizedemitted light image of the tissue.
 2. The method of claim 1, furthercomprising: inserting a probe into a patient during an operativeprocedure, wherein the generating the incident light includestransmitting the incident light from the probe, wherein the receivingthe incident light includes receiving the incident light with the probe,and wherein the receiving the emitted light includes receiving theemitted light with the probe.
 3. The method of claim 2, wherein theoperative procedure comprises imaging an internal body cavity of apatient.
 4. The method of claim 1, wherein the method comprises aselected one of a laparoscopic imaging method, an endoscopic imagingmethod, a colonoscopic imaging method, a colposcopic imaging method, anesophageal imaging, method, a pulmonary imaging method, an oral imagingmethod, and a dental imaging method.
 5. The method of claim 1, wherein:the excitation light source comprises an epi-illumination light sourcedisposed on generally the same side of the tissue as the light detector;the emitted light comprises fluorescent light associated with thetissue; the intrinsic image comprises an intrinsic epi-illuminationimage; the un-normalized emitted light image comprises an un-normalizedfluorescence epi-illumination image; and the normalized emitted lightimage comprises a normalized fluorescence epi-illumination image.
 6. Themethod of claim 5, wherein fluorescent light is generated by at leastone of a fluorescent marker and an endogenous tissue fluorescentmolecule in response to the excitation light, wherein the fluorescentmarker is administered to the tissue.
 7. The method of claim 6, whereinthe incident light is substantially at one wavelength.
 8. The method ofclaim 1, wherein: the excitation light source comprises atransillumination light source disposed on generally the opposite sideof the tissue as the light detector; the emitted light comprisesfluorescent light associated with the tissue; the intrinsic imagecomprises an intrinsic transillumination image; the un-normalizedemitted light image comprises an un-normalized fluorescencetransillumination image; and the normalized emitted light imagecomprises a normalized fluorescence transillumination image.
 9. Themethod of claim 8, wherein the fluorescent light is generated by atleast one of a fluorescent marker and an endogenous tissue fluorescentmolecule in response to the excitation light, wherein the fluorescentmarker is administered to the tissue.
 10. The method of claim 9, whereinthe incident light is substantially at one wavelength.
 11. The method ofclaim 8, wherein the transillumination light source comprises aplurality of individual transillumination light sources, wherein themethod further comprises: combining a plurality of intrinsic images togenerate the intrinsic image, wherein each one of the plurality ofintrinsic images is associated with a respective one of the plurality oftransillumination light sources, and combining a plurality ofun-normalized emitted light images to generate the un-normalized emittedlight image, wherein each one of the plurality of un-normalized emittedlight images is associated with a respective one of the plurality oftransillumination light sources.
 12. The method of claim 1, wherein: theexcitation light source comprises an epi-illumination light sourcedisposed on generally the same side of the tissue as the light detector;the emitted light comprises bioluminescence light associated with thetissue; the intrinsic image comprises an intrinsic epi-illuminationimage; the un-normalized emitted light image comprises and un-normalizedbioluminescence image; and the normalized emitted light image comprisesa normalized bioluminescence epi-illumination image.
 13. The method ofclaim 1, wherein: the excitation light source comprises atransillumination light source disposed on generally the opposite sideof the tissue as the light detector; the emitted light comprisesbioluminescence light associated with the tissue; the intrinsic imagecomprises an intrinsic transillumination image; the un-normalizedemitted light image comprises an un-normalized bioluminescence image;and the normalized emitted light image comprises a normalizedbioluminescence epi-illumination image.
 14. The method of claim 13,wherein the transillumination light source comprises a plurality ofindividual transillumination light sources, wherein the method furthercomprises: combining a plurality of intrinsic images to generate theintrinsic image, wherein each one of the plurality of intrinsic imagesis associated with a respective one of the plurality oftransillumination light sources.
 15. The method of claim 1, wherein thecombining comprises dividing the un-normalized emitted light image bythe intrinsic image.
 16. The method of claim 15, wherein the dividingcomprises dividing pixel magnitudes of pixels of the un-normalizedemitted light image by pixel magnitudes of co-registered pixels of theintrinsic image.
 17. The method of claim 1, wherein the incident lighthas a wavelength in the range of four hundred to one thousand nanometersand the emitted light also has a wavelength in the range of four hundredto one thousand nanometers, wherein the wavelength of the emitted lightis shorter than the wavelength of the excitation light.
 18. The methodof claim 1, wherein the light receiver comprises a charge coupled devicecamera.
 19. The method of claim 1, further including: generating a firstbackground image of the tissue at generally the same wavelength as theintrinsic image; generating a second background image of the tissue atgenerally the same wavelength as the un-normalized emitted light image;combining the intrinsic image and the first background image to providea noise-reduced intrinsic image; combining the un-normalized emittedlight image and the second background image to provide a noise-reducedun-normalized emitted light image; and combining the noise-reducedun-normalized emitted light image and the noise-reduced intrinsic imageto provide a noise-reduced normalized emitted light image.
 20. Themethod of claim 1, wherein the incident light includes one or morewavelengths, and wherein the intrinsic image is generated by combiningintrinsic images associated with the one or more wavelengths.
 21. Asystem for imaging a tissue, the system comprising: an excitation lightsource adapted to generate incident light including excitation light; alight receiver adapted to receive the incident light after the incidentlight has interacted with the tissue, further adapted to receive emittedlight, wherein the emitted light is emitted from the tissue, furtheradapted to generate an intrinsic image of the tissue in response to theincident light, and further adapted to generate an un-normalized emittedlight image of the tissue, and a normalization processor adapted tocombine the un-normalized emitted light image and the intrinsic image togenerate a normalized emitted light image associated with the tissue.22. The system of claim 21, further comprising: a probe; and at leastone optical fiber coupled between the probe and the excitation lightsource and between the probe and the light receiver, wherein the atleast one optical fiber is adapted to carry the incident light to theprobe, to receive the incident light with the probe after the incidentlight has interacted with the tissue, and to receive the emitted lightwith the probe.
 23. The system of claim 22, wherein the probe is adaptedto image an internal body cavity of a patient.
 24. The system of claim22, wherein probe comprises a selected one of a laparoscopic probe, anendoscopic probe, a colonoscopic probe, a colposcopic probe, anesophageal probe, a pulmonary probe, an oral probe, and a dental probe.25. The system of claim 21, wherein: the excitation light sourcecomprises an epi-illumination light source disposed on generally thesame side of the tissue as the light detector; the emitted lightcomprises fluorescent light associated with the tissue; the intrinsicimage comprises an intrinsic epi-illumination image; the un-normalizedemitted light image comprises an un-normalized fluorescenceepi-illumination image; and wherein the normalization processorcomprises a fluorescence epi-illumination normalization processoradapted to generate the normalized emitted light image as a normalizedfluorescence epi-illumination image.
 26. The system of claim 25, whereinfluorescent light is generated by at least one of a fluorescent markerand an endogenous tissue fluorescent molecule in response to theexcitation light, wherein the fluorescent marker is administered to thetissue.
 27. The system of claim 26, wherein the incident light issubstantially at one wavelength.
 28. The system of claim 21, wherein:the excitation light source comprises a transillumination light sourcedisposed on generally the opposite side of the tissue as the lightdetector; the emitted light comprises fluorescent light associated withthe tissue; the intrinsic image comprises an intrinsic transilluminationimage; the un-normalized emitted light image comprises an un-normalizedfluorescence transillumination image; and wherein the normalizationprocessor comprises a fluorescence transillumination normalizationprocessor adapted to generate the normalized emitted light image as anormalized fluorescence transillumination image.
 29. The system of claim28, wherein fluorescent light is generated by at least one of afluorescent marker and an endogenous tissue fluorescent molecule inresponse to the excitation light, wherein the fluorescent marker isadministered to the tissue.
 30. The system of claim 29, wherein theincident light is substantially at one wavelength.
 31. The system ofclaim 28, wherein the transillumination light source comprises aplurality of individual transillumination light sources, wherein thenormalization processor comprises: an intrinsic image combiningprocessor adapted to combine a plurality of intrinsic images to generatethe intrinsic image, wherein each one of the plurality of intrinsicimages is associated with a respective one of the plurality oftransillumination light sources; and an emitted image combiningprocessor adapted to combine a plurality of un-normalized emitted lightimages to generate the un-normalized emitted light image, wherein eachone of the plurality of un-normalized emitted light images is associatedwith a respective one of the plurality of transillumination lightsources.
 32. The system of claim 21, wherein: the excitation lightsource comprises an epi-illumination light source disposed on generallythe same side of the tissue as the light detector; the emitted lightcomprises bioluminescence light associated with the tissue; theintrinsic image comprises an intrinsic epi-illumination image; theun-normalized emitted light image comprises and un-normalizedbioluminescence image; and wherein the normalization processor comprisesa bioluminescence epi-illumination normalization processor adapted togenerate the normalized emitted light image as a normalizedbioluminescence epi-illumination image.
 33. The system of claim 21,wherein: the excitation light source comprises a transillumination lightsource disposed on generally the opposite side of the tissue as thelight detector; the emitted light comprises bioluminescence lightassociated with the tissue; the intrinsic image comprises an intrinsictransillumination image; the un-normalized emitted light image comprisesan un-normalized bioluminescence image; and wherein the normalizationprocessor comprises a bioluminescence transillumination normalizationprocessor adapted to generate the normalized emitted light image as anormalized bioluminescence transillumination image.
 34. The system ofclaim 33, wherein the transillumination light source comprises aplurality of individual transillumination light sources, wherein thenormalization processor comprises an intrinsic excitation imagecombining processor adapted to combine a plurality of intrinsic imagesto generate the intrinsic image, wherein each one of the plurality ofintrinsic images is associated with a respective one of the plurality oftransillumination light sources.
 35. The system of claim 21, wherein thenormalization processor is adapted to combine the un-normalized emittedlight image and the intrinsic image by dividing the un-normalizedemitted light image by the intrinsic image.
 36. The method of claim 35,wherein the normalization processor is adapted to combine theun-normalized emitted light image and the intrinsic image by dividingpixel magnitudes of pixels of the un-normalized emitted light image bypixel magnitudes of co-registered pixels of the intrinsic image.
 37. Thesystem of claim 21, wherein the incident light has a wavelength in therange of four hundred to one thousand nanometers and the emitted lightalso has a wavelength in the range off four hundred to one thousandnanometers, wherein the wavelength of the emitted light is shorter thanthe wavelength of the excitation light.
 38. The system of claim 21,wherein the light receiver comprises a charge coupled device camera. 39.The system of claim 21, further including: a first optical filtercoupled to the light detector and adapted to pass the excitation light,wherein the excitation light has a first wavelength; and a secondoptical filter coupled to the light detector and adapted to pass theemitted light, wherein the emitted light has a second wavelengthdifferent from the first wavelength.
 40. The system of claim 21, furtherincluding an optical filter coupled to the light detector and adapted topass the excitation light and the emitted light.
 41. The system of claim21, wherein the light detector is further adapted to generate a firstbackground image of the tissue at generally the same wavelength as theintrinsic image and adapted to generate a second background image of thetissue at generally the same wavelength as the un-normalized emittedlight image, and wherein the normalization processor comprises: anintrinsic image noise reduction processor adapted to combine theintrinsic image and the first background image to provide anoise-reduced intrinsic image; and an emitted image noise reductionprocessor adapted to combine the un-normalized emitted light image andthe second background image to provide a noise-reduced un-normalizedemitted light image, wherein normalization processor is further adaptedto combine the noise-reduced un-normalized emitted light image and thenoise-reduced intrinsic image to provide a noise-reduced normalizedemitted light image.
 42. The system of claim 21, wherein the incidentlight includes one or more wavelengths, and wherein the intrinsic imageis generated by combining intrinsic images associated with the one ormore wavelengths.